Culture Media Preparation Tips

Preparation of nutrient agar

Written by MicroDok

Nutrient agar is a general purpose media that supports the growth of a handful of bacteria. It is commercially available, and it comes as a powdery agar base that requires some steps to get the solidified agar required for your experiment. These steps are explained in this section. The preparation of nutrient agar is simple; and the steps outlined in this section will only serve as a guide for you.

This image shows how nutrient agar looks like after preparation

Components of nutrient agar base

The components of nutrient agar base required for nutrient agar preparation include:

  1. Agar – which is the solidifying agent
  2. Peptone – which provides nitrogen source
  3. Yeast/beef extract – which provides carbohydrates, vitamins
  4. Sodium chloride – which provides a physiological state akin to that of the living system
  5. pH – which is usually adjusted to 6.8 at 25 °C (or 77 °F).

NOTE: The amount of powdered agar base to weigh for the preparation of any culture media of your choice varies from one culture media to another, since manufacturers instructions usually vary from one type of media to another. The important point is to always follow manufacturers instruction of each media (as outlined on the side of the culture media bottle or container); and keeping to the steps outlined here, you will make a good culture media for your experiment.

All these components are already present in the nutrient agar base that you purchased commercially; and so you do not need to worry on how to obtain and mix them. All you need now is to follow the following steps and get your nutrient agar prepared. This also apply to some other commercially available culture media base.

MATERIALS

You require these materials to prepare your nutrient agar: nutrient agar powder (usually comes in 500 g), autoclave, conical flask, measuring cylinder, beaker, stirring rod, Bunsen burner, incubator, refrigerator, wire gauze, spatula, weighing balance, timer, cotton wool, aluminium foil, distilled water, Petri dish

STEP BY STEP PROTOCOL TO PREPARE NUTRIENT AGAR

  1. Weigh out 28 g of nutrient agar powder using the weighing balance. NOTE: 28 g is for the preparation of 1000 ml or 1 litre of nutrient agar (which will only give you 50 plates of nutrient agar, since each prepared plate should contain about 20 ml of molten agar). In another section, you will see how to do the simple calculation to prepare any number of plates of nutrient agar or any other bacteriological agar of your choice.
  2. Suspend the 28 g of nutrient agar powder in 1 litre (1000 ml) of distilled water in a conical flask.
  3. Mix the solution by stirring to dissolve the agar.
  4. Bring the mixture to boil, by mild boiling of the mixture over a Bunsen burner flame. This helps to dissolve the agar completely. Monitor the boiling process closely in order to avoid charring the agar.
  5. Transfer the conical flask containing the boiled/mixed nutrient agar suspension to the autoclave.
  6. Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
  7. At the end of sterilization, allow the autoclave to return to normal (zero point) before opening. Otherwise the pressure built up in the autoclave will affect the quality and quantity of the prepared molten medium. More so, you may be affected by the steam from the autoclave due to the high pressure built up in the autoclave.
  8. Allow molten medium to cool to about 45-50 degrees Celsius.
  9. Pour prepared molten medium into sterile Petri dish plates.
  10. Allow the poured plates on the bench to solidify.
  11. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  12. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  13. Absence of colony on the plate means that your sterilization is good.
  14. You can now use your prepared nutrient agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.

REFERENCES

  1. Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. Part 2 . Cambridge University Press, UK.

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MicroDok

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